Fig. 3: Fixing DHp domain rotation to tune kinase activity and isolate strong phosphatases.
From: Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles
a Fusion of GCN4 leucine zipper to the N-terminus of EnvZ truncated between residues 212 and 221, thus connecting to the DHp domain and fixing the rotation of its alpha helices41. The box on the right shows the sequence of the first 37–46 amino acids of each variant. b Kinase and phosphatase activity assays for each rotationally locked variant (same experimental design as in Fig. 2e–f). EnvZm2, a kinase-biased variant, establishes a baseline of phosphorylated OmpR for testing dephosphorylation. EnvZm3t# have mutation 'm3' (N343K), which knocks out kinase activity. c Maximum fold-change in output expression induced by each variant mapped to the putative rotational conformation of the DHp domain, assuming 100° of rotation for each amino acid truncated between GCN4 and the DHp domain and setting EnvZt1 to 0∘. All data were measured by flow cytometry at 48 hours post transfection in HEK-293FT cells. All error bars represent the mean ± s.d. of measurements from three experimental repeats. Source data are provided as a Source Data file.
